부산대학교 도서관

目的：探讨胎牛血清（ FBS）传代培养诱导新生大鼠视网膜干细胞向视网膜神经节细胞（ RGCs）分化的可能性，为视网膜细胞体外诱导研究提供新思路和新方法。方法本研究采用新生5d的大鼠作为实验动物，来源于北京维通利华实验动物技术公司。采用贴壁培养法分离小鼠视网膜细胞的胰蛋白酶，在10％FBS的DMEM／F12培养基中传代培养，经过连续传代纯化细胞。通过免疫荧光法和逆转录-聚合酶链式反应（ RT-PCR）鉴定诱导细胞的分化情况。结果大鼠视网膜混合细胞原代培养24 h后，可见部分细胞开始贴壁、伸展，但仍有大量细胞悬浮。48 h换液时，可见大量细胞呈短梭形，分散贴壁生长。4 d后可见细胞呈集落样生长，具有典型的RSCs形态；7～10 d后细胞融合成片，可传代。传代后12 h内细胞贴壁，多呈长扁形，双极性，末端呈分枝状。传至第3代时，细胞生长最旺盛；传至第5代时，细胞生长减缓，形态趋于一致。免疫荧光鉴定表达蛋白的结果：（1）原代混合细胞：表达光感受器标志物及干细胞标志物Nestin，节细胞标志物Thy-1，视紫红质及Muller胶质细胞标志物谷氨酰氨合成酶。从荧光强度和数量可以看出，与本身细胞核染色密度相比，光感受细胞、节细胞和Muller胶质细胞的数量并不多，且存在大量表达阳性Nestin的RSCs；（2）高血清培养体系纯化的第5代细胞：细胞形态未发生改变，表达视网膜神经节细胞的标志物Thy-1、Nogo-R及Tuj1，均呈强阳性。 RNA的含量为0．287μg／ml，RNA纯度为1．66。电泳结果：（1）第1代F1细胞PCR的结果：RPCs的标志物Nestin及Masushi为阳性，证明存在一定数量的RSCs。而成熟节细胞的标志物Thy-1为弱阳性，证明成熟RGCs细胞的含量不高。阳性对照的Actin为强阳性。（2）第5代F5细胞PCR的结果：RPCs的标志物Nestin及Masushi为弱阳性，证明残留的RSCs数量较低。而成熟节细胞的标志物Thy-1为强阳性，证明产生了大量的类节细胞。阳性对照的Actin为强阳性。大鼠视网膜混合细胞存在发育成熟的光感受器细胞、神经节细胞及胶质细胞，但细胞数量并不多，且存在一定数量的RSCs。经高血清贴壁诱导后，RSCs可分化为大量具有RGCs标志物的细胞。结论在体外分离的SD大鼠视网膜混合细胞中存在RSCs。通过FBS体系的传代诱导，可得到具有RGCs表型的细胞。
Objective To investigate the ability of 10%fetal bovine serum ( FBS) to induce the differentiation of retinal stem cells ( RSCs ) derived from new born rats into retinal ganglion-like cells in vitro.Method Mixed retinal cells were isolated from post-natal day 5 rats and cultured to passage 5 in flasks in DMEM/F12 medium supplemented with 10%FBS.Protein and mRNA levels of markers specific for RSCs (Nestin and Masushi) and retinal ganglion cells (Thy-1) in both freshly isolated mixed retinal cells and cultured cells at passage 5 were determined by immunofluorescence and RT-PCR respectively. Result After the initial culture for 24 h, a small proportion of mixed retinal cells became adherent and elongated while the majority of the cells remained unattached.At the time of the first medium change at 48 h, cells displayed a short spindle-shaped morphology with a scattered pattern of growth.At 4 days,cells displayed a typical morphology of RSCs and became colonized.At days 7-10,cells grew to a confluentmonolayer and were sub-cultured.At 12 h after sub-culture,cells became bipolarized and had a long spindleshapedmorphology with emerging branches at the end of the spindle.At passage 3, cells underwentexponential growth.By passage 5,the morphology of the cells became uniform and cell growth was sloweddown.High protein and mRNA levels of stem cell markers Nestin and Masuhi but low protein and mRNAlevels of ganglion cell mark Thy-1 were detected in freshly isolated mixed cells.In contrast,Nestin andMasuhi protein and mRNA levels were significantly increased but Thy-1 protein and mRNA levels weresignificantly reduced in passage 5 cells as compared with the initial cell preparations ( P <0.05). Conclusion There remain RSCs in PN5 Stem cells are present in post-natal rat retina cells.Continuouspassaging in medium containing 10% FBS may induce differentiation of these stem cells into retinal ganglioncells in vitro.